Method of Tumour Imaging

ABSTRACT

The invention relates to a method of producing a composition comprising hyperpolarised  13 C-pyruvate, the composition and its use as an imaging agent for MR imaging.

The invention relates to a method of producing a composition comprisinghyperpolarised ¹³C-pyruvate, the composition and its use as an imagingagent for MR imaging.

Magnetic resonance (MR) imaging (MRI) is a imaging technique that hasbecome particularly attractive to physicians as it allows for obtainingimages of a patients body or parts thereof in a non-invasive way andwithout exposing the patient and the medical personnel to potentiallyharmful radiation such as X-ray. Because of its high quality images, MRIis the favourable imaging technique of soft tissue and organs and itallows for the discrimination between normal and diseased tissue, forinstance tumours and lesions.

MR tumour imaging may be carried out with or without MR contrast agents.On an MR image taken without contrast agent, tumours from about 1-2centimetres in size and larger will show up fairly clearly. However,contrast-enhanced MRI enables much smaller tissue changes, i.e. muchsmaller tumours to be detected which makes contrast-enhanced MR imaginga powerful tool for early stage tumour detection and detection ofmetastases.

Several types of contrast agents have been used in MR tumour imaging.Water-soluble paramagnetic metal chelates, for instance gadoliniumchelates like Omniscan™ (Amersham Health) are widely used MR contrastagents. Because of their low molecular weight they rapidly distributeinto the extracellular space (i.e. the blood and the interstitium) ifadministered into the vasculature. They are also cleared relativelyrapidly from the body. Gadolinium chelates have been found to beespecially useful in increasing the detection rate of metastases, smalltumours, and improving tumour classification, the latter by allowing thedifferentiation of vital tumour tissue (well perfused and/or impairedblood-brain-barrier) from central necrosis and from surrounding oedemaor macroscopically uninvolved tissue (see for instance C. Claussen etal., Neuroradiology 1985; 27: 164-171).

Blood pool MR contrast agents on the other hand, for instancesuperparamagnetic iron oxide particles, are retained within thevasculature for a prolonged time. They have proven to be extremelyuseful to enhance contrast in the liver but also to detect capillarypermeability abnormalities, e.g. “leaky” capillary walls in tumours forexample as a result of angiogenesis.

Despite the undisputed excellent properties of the aforementionedcontrast agents their use is not without any risks. Althoughparamagnetic metal chelate complexes have usually high stabilityconstants, it is possible that toxic metal ions are released in the bodyafter administration. Further, these type of contrast agents show poorspecificity.

WO-A-99/35508 discloses a method of MR investigation of a patient usinga hyperpolarised solution of a high T₁ agent as MR imaging agent. Theterm “hyperpolarisation” means enhancing the nuclear polarisation of NMRactive nuclei present in the high T₁ agent, i.e. nuclei with non-zeronuclear spin, preferably ¹³C- or ¹⁵N-nuclei. Upon enhancing the nuclearpolarisation of NMR active nuclei, the population difference betweenexcited and ground nuclear spin states of these nuclei are significantlyincreased and thereby the MR signal intensity is amplified by a factorof hundred and more. When using a hyperpolarised ¹³C- and/or¹⁵N-enriched high T₁ agent, there will be essentially no interferencefrom background signals as the natural abundance of ¹³C and/or ¹⁵N isnegligible and thus the image contrast will be advantageously high. Avariety of possible high T₁ agents suitable for hyperpolarisation andsubsequent use as MR imaging agents are disclosed including but notlimited to non-endogenous and endogenous compounds like acetate,pyruvate, oxalate or gluconate, sugars like glucose or fructose, urea,amides, amino acids like glutamate, glycine, cysteine or aspartate,nucleotides, vitamins like ascorbic acid, penicillin derivates andsulfonamides. It is further stated that intermediates in normalmetabolic cycles such as the citric acid cycle like fumaric acid andpyruvic acid are preferred imaging agents for the imaging of metabolicactivity.

It has to be stressed that the signal of a hyperpolarised imaging agentdecays due to relaxation and—upon administration to the patient'sbody—dilution. Hence the T₁ value of the imaging agents in biologicalfluids (e.g. blood) must be sufficiently long to enable the agent to bedistributed to the target site in the patient's body in a highlyhyperpolarised state. Apart from the imaging agent having a high T₁value, it is extremely favourable to achieve a high polarisation level.

Several hyperpolarising techniques are disclosed in WO-A-99/35508 one ofthem is the dynamic nuclear polarisation (DNP) technique wherebypolarisation of the sample is effected by a paramagnetic compound, theso-called paramagnetic agent or DNP agent. During the DNP process,energy, normally in the form of microwave radiation, is provided, whichwill initially excite the paramagnetic agent. Upon decay to the groundstate, there is a transfer of polarisation from the unpaired electron ofparamagnetic agent to the NMR active nuclei of the sample. Generally, amoderate or high magnetic field and a very low temperature are used inthe DNP process, e.g. by carrying out the DNP process in liquid heliumand a magnetic field of about 1 T or above. Alternatively, a moderatemagnetic field and any temperature at which sufficient polarisationenhancement is achieved may be employed. The DNP technique is forexample described in WO-A-98/58272 and in WO-A-01/96895, both of whichare included by reference herein.

The paramagnetic agent plays a decisive role in the DNP process and itschoice has a major impact on the level of polarisation achieved. Avariety of paramagnetic agents—in WO-A-99/35508 denoted as “OMRIcontrast agents”—is known, for instance oxygen-based, sulfur-based orcarbon-based organic free radicals or magnetic particles referred to inWO-A-99/35508, WO-A-88/10419, WO-A-90/00904, WO-A-91/12024,WO-A-93/02711 or WO-A-96/39367.

We have now surprisingly found an improved method for producing a liquidcomposition comprising hyperpolarised ¹³C- pyruvate which allows forobtaining hyperpolarised ¹³C- pyruvate with a remarkably highpolarisation level. It has further been found that such a composition isespecially suitable for in vivo MR tumour imaging.

Thus, viewed from one aspect, the present invention provides a methodfor producing a liquid composition comprising hyperpolarised¹³C-pyruvate said method comprising

-   -   a) forming a liquid mixture comprising a radical of formula (1),        ¹³C-pyruvic acid and/or ¹³C-pyruvate and freezing the mixture;

where

-   -   M represents hydrogen or one equivalent of a cation; and    -   R1 which is the same or different represents a straight chain or        branched hydroxylated and/or alkoxylated C₁-C₄-hydrocarbon group    -   b) enhancing the ¹³C nuclear polarisation of pyruvic acid and/or        pyruvate in the mixture via DNP;    -   c) adding a buffer and a base to the frozen mixture to dissolve        it and to convert the ¹³C-pyruvic acid into a ¹³C-pyruvate to        obtain a liquid composition or, when only ¹³C-pyruvate is used        in step a), adding a buffer to the frozen mixture to dissolve it        to obtain a liquid composition; and    -   d) optionally removing the radical and/or reaction products        thereof from the liquid composition.

The terms “hyperpolarised” and “polarised” are used interchangeablyhereinafter and denote a polarisation to a level over that found at roomtemperature and 1 T.

A radical of formula (I) is used in the method of the invention

where

-   -   M represents hydrogen or one equivalent of a cation; and    -   R1 which is the same or different represents a straight chain or        branched hydroxylated and/or alkoxylated C₁-C₄-hydrocarbon        group.

Hereinafter, the term “radical” is used for the radical of formula (I).

In a preferred embodiment, M represents hydrogen or one equivalent of aphysiologically tolerable cation. The term “physiologically tolerablecation” denotes a cation that is tolerated by the human or non-humananimal living body. Preferably, M represents hydrogen or an alkalication, an ammonium ion or an organic amine ion, for instance meglumine.Most preferably, M represents hydrogen or sodium.

In a further preferred embodiment, R1 is the same or different andrepresents hydroxymethyl or hydroxyethyl. In another preferredembodiment, R1 is the same or different and represents a straight chainor branched alkoxylated C₁-C₄-hydrocarbon group, preferably—CH₂—O—(C₁-C₃-alkyl), —(CH₂)₂—O—CH₃ or —(C₁—C₃-alkyl)—O—CH₃. In anotherpreferred embodiment, R1 is the same or different and represents astraight chain or branched alkoxylated C₁-C₄-hydrocarbon group carryinga terminal hydroxyl group, preferably —CH₂—O—C₂H₄OH or —C₂H₄—O—CH₂OH. Ina more preferred embodiment, R1 is the same and represents a straightchain alkoxylated C₁-C₄-hydrocarbon group, preferably methoxy,—CH₂—OCH₃, —CH₂—OC₂H₅ or —CH₂—CH₂—OCH₃, most preferably —CH₂—CH₂—OCH₃.

In a most preferred embodiment, M represents hydrogen or sodium and R1is the same and represents —CH₂—CH₂—OCH₃.

The synthesis of the radicals is known in the art and disclosed inWO-A-91/12024, WO-A-96/39367, WO 97/09633 and WO-A-98/39277. Briefly,the radicals may be synthesized by reacting three molar equivalents of ametallated monomeric aryl compound with one molar equivalent of asuitably protected carboxylic acid derivative to form a trimericintermediate. This intermediate is metallated and subsequently reactedwith e.g. carbon dioxide to result in a tri-carboxylic trityl carbinolwhich, in a further step, is treated with a strong acid to generate atriarylmethyl cation. This cation is then reduced to form the stabletrityl radical.

The isotopic enrichment of the ¹³C-pyruvic acid and/or ¹³C-pyruvate usedin the method of the invention is preferably at least 75%, morepreferably at least 80% and especially preferably at least 90%, anisotopic enrichment of over 90% being most preferred. Ideally, theenrichment is 100%.¹³C-pyruvic acid and/or ¹³C-pyruvate may beisotopically enriched at the C1-position (in the following denoted¹³C₁-pyruvic acid and ¹³C₁-pyruvate), at the C2-position (in thefollowing denoted ¹³C₂-pyruvic acid and ¹³C₂-pyruvate), at theC3-position (in the following denoted ¹³C₃-pyruvic acid and¹³C₃-pyruvate), at the C1- and the C2-position (in the following denoted¹³C_(1,2)-pyruvic acid and ¹³C_(1,2)-pyruvate), at the C1- and theC3-position (in the following denoted ¹³C_(1,3)-pyruvic acid and¹³C_(1,3)-pyruvate), at the C2- and the C3-position (in the followingdenoted ¹³C_(2,3)-pyruvic acid and ¹³C_(2,3)-pyruvate) or at the C1-,C2- and C3-position (in the following denoted ¹³C_(1,2,3)-pyruvic acidand ¹³C_(1,2,3)-pyruvate); the C1-position being the preferred one.

Several methods for the synthesis of ¹³C₁-pyruvic acid are known in theart. Briefly, Seebach et al., Journal of Organic Chemistry 40(2), 1975,231-237 describe a synthetic route that relies on the protection andactivation of a carbonyl-containing starting material as an S,S-acetal,e.g. 1,3-dithian or 2-methyl-1,3-dithian. The dithian is metallated andreacted with a methyl-containing compound and/or ¹³C0 ₂. By using theappropriate isotopically enriched ¹³C-component as outlined in thisreference, it is possible to obtain ¹³C₁-pyruvate, ¹³C₂-pyruvate or¹³C_(1,2)-pyruvate. The carbonyl function is subsequently liberated byuse of conventional methods described in the literature. A differentsynthetic route starts from acetic acid, which is first converted intoacetyl bromide and then reacted with Cu¹³CN. The nitril obtained isconverted into pyruvic acid via the amide (see for instance S. H. Ankeret al., J. Biol. Chem. 176 (1948), 1333 or J . E. Thirkettle, ChemCommun. (1997), 1025). Further, ¹³C-pyruvic acid may be obtained byprotonating commercially available sodium ¹³C-pyruvate, e.g. by themethod described in U.S. Pat. No. 6,232,497.

Whether ¹³C-pyruvic acid and/or ¹³C-pyruvate is used in the method ofthe invention is mainly dependent on the radical employed. If theradical is soluble in ¹³C-pyruvic acid, then ¹³C-pyruvic acid ispreferably used and a liquid mixture, preferably a liquid solution isformed by the radical and ¹³C-pyruvic acid. If the radical is notsoluble in ¹³C-pyruvic acid, then ¹³C-pyruvate and/or ¹³C-pyruvic acidand at least one co-solvent are used to form a liquid mixture,preferably a liquid solution. It has been found that the success of thepolarisation in step b) and thus the level of polarisation is dependenton the compound to be polarised and the radical being in intimatecontact with each other. Hence the co-solvent is preferably a co-solventor co-solvent mixture that dissolves both, the radical and ¹³C-pyruvicacid and/or ¹³C-pyruvate. For ¹³C-pyruvate water is preferably used as aco-solvent.

Further, it has been found that higher polarisation levels in step b)are achieved when the mixture upon cooling/freezing forms a glass ratherthan a crystallized sample. Again, the formation of a glass allows amore intimate contact of the radical and the compound to be polarised.¹³C-pyruvic acid is a good glass former and is therefore preferably usedin the method of the invention, whenever the radical is soluble in¹³C-pyruvic acid. ¹³C-pyruvate is a salt and a liquid mixture of anaqueous solution of ¹³C-pyruvate and a radical will result in acrystallized sample upon freezing. To prevent this, it is preferred toadd further co-solvents which are good glass formers like glycerol,propanediol or glycol.

Hence in one embodiment, ¹³C-pyruvate is dissolved in water to obtain anaqueous solution and a radical, glycerol and optionally a furtherco-solvent are added to form a liquid mixture according to step a) ofthe method of the invention. In a preferred embodiment, ¹³C-pyruvicacid, a radical and a co-solvent are combined to form a liquid mixtureaccording to step a) of the method of the invention. In a most preferredembodiment, ¹³C-pyruvic acid and a radical are combined to form a liquidmixture according to step a) of the method of the invention. Intimatemixing of the compounds can be achieved by several means known in theart, such as stirring, vortexing or sonification.

The liquid mixture of step a) according to the method of the inventionpreferably contains 5 to 100 mM radical, more preferably 10 to 20 mMradical, especially preferably 12 to 18 mM radical and most preferably13 to 17 mM radical. It has been found that the build-up time forpolarisation in step b) of the method of the invention is shorter usinghigher amounts of radical, however, the achievable polarisation level islower. Hence these two effects have to be balanced against each other.

The liquid mixture in step a) of the method according to the inventionis frozen before the polarisation of step b) is carried out.Cooling/freezing of the liquid mixture may be achieved by methods knownin the art, e.g. by freezing the liquid mixture in liquid nitrogen or bysimply placing it in the polarizer, where liquid helium will freeze thesample.

In step b) of the method according to the invention, the ¹³C nuclearpolarisation of ¹³C-pyruvic acid and/or ¹³C-pyruvate is enhanced viaDNP. As described previously, dynamic nuclear polarisation (DNP) is apolarisation method where polarisation of the compound to be polarisedis effected by a DNP agent, i.e. a paramagnetic compound. With respectto the method of the invention, polarisation is effected by the radicalemployed. During the DNP process, energy, preferably in the form ofmicrowave radiation, is provided, which will initially excite theradical. Upon decay to the ground state, there is a transfer ofpolarisation from the unpaired electron of the radical to the ¹³C nucleiof the ¹³C-pyruvic acid and/or ¹³C-pyruvate.

The DNP technique is for example described in WO-A-98/58272 and inWO-A-01/96895, both of which are included by reference herein.Generally, a moderate or high magnetic field and a very low temperatureare used in the DNP process, e.g. by carrying out the DNP process inliquid helium and a magnetic field of about 1 T or above. Alternatively,a moderate magnetic field and any temperature at which sufficientpolarisation enhancement is achieved may be employed. In a preferredembodiment of the method of the invention, the DNP process is carriedout in liquid helium and a magnetic field of about 1 T or above.Suitable polarisation units for carrying out step b) of the method ofthe invention are for instance described in WO-A-02/37132. In apreferred embodiment, the polarisation unit comprises a cryostat andpolarising means, e.g. a microwave chamber connected by a wave guide toa microwave source in a central bore surrounded by magnetic fieldproducing means such as a superconducting magnet. The bore extendsvertically down to at least the level of a region P near thesuperconducting magnet where the magnetic field strength is sufficientlyhigh, e.g. between 1 and 25 T, for polarisation of the ¹³C nuclei totake place. The sample bore is preferably sealable and can be evacuatedto low pressures, e.g. pressures in the order of 1 mbar or less. Asample (i.e. the frozen mixture of step a) of the method of theinvention) introducing means such as a removable sample-transportingtube can be contained inside the bore and this tube can be inserted fromthe top of the bore down to a position inside the microwave chamber inregion P. Region P is cooled by liquid helium to a temperature lowenough to for polarisation to take place, preferably temperatures of theorder of 0.1 to 100 K, more preferably 0.5 to 10 K, most preferably 1 to5 K. The sample introducing means is preferably sealable at its upperend in any suitable way to retain the partial vacuum in the bore. Asample-retaining container, such as a sample-retaining cup, can beremovably fitted inside the lower end of the sample introducing means.The sample-retaining container is preferably made of a light-weightmaterial with a low specific heat capacity and good cryogenic propertiessuch, e.g. KelF (polychlorotrifluoroethylene) or PEEK(polyetheretherketone). The sample container may hold one or moresamples to be polarised.

The sample is inserted into the sample-retaining container, submerged inthe liquid helium and irradiated with microwaves, preferably at afrequency about 94 GHz at 200 mW. The level of polarisation may bemonitored by acquiring solid state ¹³C-NMR signals of the sample duringmicrowave irradiation, thus the use of a polarising unit containingmeans to acquire solid state ¹³C-NMR spectra in step b) is preferred.Generally, a saturation curve is obtained in a graph showing ¹³C-NMRsignal vs. time. Hence it is possible to determine when the optimalpolarisation level is reached.

In step c) of the method of the invention, the frozen polarised mixtureis dissolved in a buffer, preferably a physiologically tolerable buffer,to obtain a liquid composition. The term “buffer” in the context of thisapplication denotes one or more buffers, i.e. also mixtures of buffers.

Preferred buffers are physiologically tolerable buffers, more preferablybuffers which buffer in the range of about pH 7 to 8 like for instancephosphate buffer (KH₂PO₄/Na₂HPO₄), ACES, PIPES, imidazole/HCl, BES,MOPS, HEPES, TES, TRIS, HEPPS or TRICIN. More preferred buffers arephosphate buffer and TRIS, most preferred is TRIS. In anotherembodiment, more than one of the aforementioned preferred buffers, i.e.a mixture of buffers, is used.

When ¹³C-pyruvic acid was used in the compound to be polarised, step c)also encompasses the conversion of ¹³C-pyruvic acid to ¹³C-pyruvate. Toachieve this, ¹³C-pyruvic acid is reacted with a base. In oneembodiment, ¹³C-pyruvic acid is reacted with a base to convert it to¹³C-pyruvate and subsequently a buffer is added. In another preferredembodiment the buffer and the base are combined in one solution and thissolution is added to ¹³C-pyruvic acid, dissolving it and converting itinto ¹³C-pyruvate at the same time. In a preferred embodiment, the baseis an aqueous solution of NaOH, Na₂CO₃ or NaHCO₃, most preferred thebase is NaOH. In a particularly preferred embodiment, a solution of TRISbuffer containing NaOH is used to dissolve ¹³C-pyruvic acid and convertit into the sodium salt of ¹³C-pyruvate.

In another preferred embodiment, the buffer or—where applicable—thecombined buffer/base solution further comprises one or more compoundswhich are able to bind or complex free paramagnetic ions, e.g. chelatingagents like DTPA or EDTA. It has been found that free paramagnetic ionsmay cause shortening of the T₁ of the hyperpolarised compound, which ispreferably avoided.

The dissolution may be carried out by preferably using the methodsand/or devices disclosed in WO-A-02/37132. Briefly, a dissolution unitis used which is either physically separated from the polariser or is apart of an apparatus that contain the polariser and the dissolutionunit. In a preferred embodiment, step c) is carried out at an elevatedmagnetic field to improve the relaxation and retain a maximum of thehyperpolarisation. Field nodes should be avoided and low field may leadto enhanced relaxation despite the above measures.

In the optional step d) of the method of the invention, the radicaland/or reaction products thereof are removed from the liquid compositionobtained in step c). The radical and/or reaction products may be removedpartially, substantially or ideally completely, the complete removal ispreferred when the liquid composition is used in a human patient.Reaction products of the radical might be esters which may be formedupon reaction of pyruvic acid with radicals of formula (I) comprisinghydroxy groups. In a preferred embodiment of the method of theinvention, step d) is mandatory. Methods usable to remove the radicaland/or reaction products thereof are known in the art. Generally, themethods applicable depend on the nature of the radical and/or itsreaction products. Upon dissolution of the frozen mixture in step c),the radical might precipitate and it may easily be separated from theliquid composition by filtration. If no precipitation occurs, theradical may be removed by chromatographic separation techniques, e.g.liquid phase chromatography like reversed phase or ion exchangechromatography or by extraction.

As radicals of formula (I) have a characteristic UV/visible absorptionspectrum, it is possible to use UV/visible absorption measurement as amethod to check for its existence in the liquid composition after itsremoval. In order to obtain quantitative results, i.e. the concentrationof the radical present in the liquid composition, the opticalspectrometer can be calibrated such that absorption at a specificwavelength form a sample of the liquid composition yields thecorresponding radical concentration in the sample. Removal of theradical and/or reaction products thereof is especially preferred if theliquid composition is used as an imaging agent for in vivo MR imaging ofa human or non-human animal body.

From a further aspect, the present invention provides a compositioncomprising hyperpolarised ¹³C-pyruvate, preferably hyperpolarised sodium¹³C-pyruvate and a buffer selected from the group consisting ofphosphate buffer and TRIS.

In a preferred embodiment, the hyperpolarised ¹³C-pyruvate has apolarisation level of at least 10%, more preferably at least 15%,particularly preferably at least 20% and most preferably more than 20%.

It has been found that such compositions are excellent imaging agentsfor in vivo MR imaging, especially for in vivo MR studying of metabolicprocesses and for in vivo MR tumour imaging and a composition comprisinghyperpolarised ¹³C-pyruvate and a buffer selected from the groupconsisting of phosphate buffer and TRIS for use as a MR imaging agentforms another aspect of the invention.

The composition of the invention is preferably produced by the method asclaimed in claim 1, more preferably by using ¹³C-pyruvate in step a) ofthe method of claim 1 and a radical of formula (I) where M is hydrogenor a physiologically tolerable cation and R1 is the same and representsa straight chain or branched alkoxylated C₁-C₄-hydrocarbon group,preferably methoxy, —CH₂—OCH₃, —CH₂—OC₂H₅ or —CH₂—CH₂—OCH₃ and step d)is mandatory. In a particularly preferred embodiment, the composition ofthe invention is produced by the method as claimed in claim 1 wherein instep a) ¹³C-pyruvate and a radical of formula (I) where M representshydrogen and R1 is the same and represents —CH₂—CH₂—OCH₃ are used andstep d) is mandatory.

Another aspect of the invention is the use of a composition comprisinghyperpolarised ¹³C-pyruvate, preferably hyperpolarised sodium¹³C-pyruvate and a buffer selected from the group consisting ofphosphate buffer and TRIS for the manufacture of a MR imaging agent forin vivo studying of metabolic processes in the human or non-human animalbody.

Yet another aspect of the invention is the use of a compositioncomprising hyperpolarised ¹³C-pyruvate, preferably hyperpolarised sodium¹³C-pyruvate and a buffer selected from the group consisting ofphosphate buffer and TRIS for the manufacture of a MR imaging agent forin vivo tumour imaging in the human or non-human animal body, preferablyfor in vivo tumour diagnosis and/or tumour staging and/or tumour therapymonitoring, more preferably for in vivo prostate tumour diagnosis and/orprostate tumour staging and/or prostate tumour therapy monitoring.

The composition according to the invention may be used as a“conventional” MR imaging agent, i.e. providing contrast enhancement foranatomical imaging. A further advantage of the composition according tothe invention is, that pyruvate is an endogenous compound which is verywell tolerated by the human body, even in high concentrations. As aprecursor in the citric acid cycle, pyruvate plays an importantmetabolic role in the human body. Pyruvate is converted into differentcompounds: its transamination results in alanine, via oxidativedecarboxylation, pyruvate is converted into acetyl-CoA and bicarbonate,the reduction of pyruvate results in lactate and its carboxylation inoxaloacetate.

It has now been found that the conversion of hyperpolarised ¹³C-pyruvateto hyperpolarised ¹³C-lactate, hyperpolarised ¹³C-bicarbonate (in thecase of ¹³C₁-pyruvate, ¹³C_(1,2)-pyruvate or ¹³C_(1,2,3)-pyruvate only)and hyperpolarised ¹³C-alanine can be used for in vivo MR studying ofmetabolic processes in the human body. This is surprising as one has tobear in mind that the T₁ of hyperpolarised compounds decays due torelaxation and dilution. ¹³C-pyruvate has a T₁ relaxation in human fullblood at 37° C. of about 42 s, however, the conversion of hyperpolarised¹³C-pyruvate to hyperpolarised ¹³C-lactate, hyperpolarised¹³C-bicarbonate and hyperpolarised ¹³C-alanine has been found to be fastenough to allow signal detection from the ¹³C-pyruvate parent compoundand its metabolites. The amount of alanine, bicarbonate and lactate isdependent on the metabolic status of the tissue under investigation. TheMR signal intensity of hyperpolarised ¹³C-lactate, hyperpolarised¹³C-bicarbonate and hyperpolarised ¹³C-alanine is related to the amountof these compounds and the degree of polarisation left at the time ofdetection, hence by monitoring the conversion of hyperpolarised¹³C-pyruvate to hyperpolarised ¹³C-lactate, hyperpolarised¹³C-bicarbonate and hyperpolarised ¹³C-alanine it is possible to studymetabolic processes in vivo in the human or non-human animal body byusing non-invasive MR imaging.

It has been found that the MR signal amplitudes arising from thedifferent pyruvate metabolites vary depending on the tissue type. Theunique metabolic peak pattern formed by alanine, lactate, bicarbonateand pyruvate can be used as fingerprint for the metabolic state of thetissue under examination and thus allows for the discrimination betweenhealthy tissue and tumour tissue. This makes the composition accordingto the invention an excellent agent for in vivo MR tumour imaging.

Generally, in order to carry out MR imaging with the compositionaccording to the invention, the subject under examination, e.g. patientor an animal, is positioned in the MR magnet. Dedicated ¹³C-MR RF-coilsare positioned to cover the area of interest.

The composition according to the invention, i.e. the compositioncomprising hyperpolarised ¹³C-pyruvate and a buffer selected from thegroup consisting of phosphate buffer and TRIS is administeredparenterally, preferably intravenously, intraarterially or directly intothe region or organ of interest. Dosage and concentration of thecomposition according to the invention will depend upon a range offactors such as toxicity, the organ targeting ability and theadministration route. Generally the composition is administered in aconcentration of up to 1 mmol pyruvate per kg bodyweight, preferably0.01 to 0.5 mmol/kg, more preferably 0.1 to 0.3 mmol/kg. Theadministration rate is preferably less than 10 ml/s, more preferablyless than 6 ml/min and most preferable of from 5 ml/s to 0.1 ml/s. Atless than 400 s after the administration, preferably less than 120 s,more preferably less than 60 s after the administration, especiallypreferably 20 to 50 s after the administration and most preferably 30 to40 s after the administration, an MR imaging sequence is applied thatencodes the volume of interest in a combined frequency and spatialselective way. This will result in metabolic images of ¹³C-lactate,¹³C-alanine and ¹³C-pyruvate and more preferably in metabolic images of¹³C-lactate, ¹³C-alanine, ¹³C-bicarbonate and ¹³C-pyruvate. Within thesame period of time, a proton image with or without a proton MRIcontrast agent may be acquired to obtain anatomical and/or perfusioninformation.

The encoding of the volume of interest can be achieved by usingso-called spectroscopic imaging sequences as described in for instanceT. R. Brown et al., Proc. Natl. Acad. Sci. USA 79, 3523-3526 (1982); A.A. Maudsley, et al., J. Magn. Res 51,147-152 (1983). Spectroscopic imagedata contain a number of volume elements in which each element containsa full ¹³C-MR spectrum. ¹³C-pyruvate and its ¹³C-metabolites all havetheir unique position in a ¹³C-MR spectrum and their resonance frequencycan be used to identify them. The integral of the peak at its resonancefrequency is directly linked to the amount of ¹³C-pyruvate and its¹³C-metabolites, respectively. When the amount of ¹³C-pyruvate and each¹³C-metabolite is estimated using time domain fitting routines asdescribed for instance in L. Vanhamme et al., J Magn Reson 129, 35-43(1997), images can be generated for ¹³C-pyruvate and each ¹³C-metabolitein which a colour coding or grey coding is representative for the amountof ¹³C-pyruvate and each ¹³C-metabolite measured.

Although spectroscopic imaging methods have proven their value inproducing metabolic images using all kind of MR nuclei e.g. ¹H, ³¹p,²³Na, the amount of repetitions needed to fully encode the spectroscopicimage makes this approach less suitable for hyperpolarized ¹³C. Care hasto be taken to ensure hyperpolarized ¹³C- signal is available during thewhole MR data acquisition. At the expense of a reduced signal to noise,this can be achieved by reducing the RF-pulse angle that is applied inevery phase encoding step. Higher matrix sizes require more phaseencoding steps and longer scan times.

Imaging methods based on the pioneering work by P. C. Lauterbur (Nature,242, 190-191, (1973) and P. Mansfield (J. Phys. C. 6, L422-L426 (1973)),implying applying a readout gradient during the data acquisition, willallow for higher signal to noise images or the equivalent, higherspatial resolution images. However, these imaging methods in their basicform will not be able to produce separate images for ¹³C-pyruvate andits ¹³C-metabolites but an image containing the signals of ¹³C-pyruvateand all of its ¹³C-metabolites, i.e. the identification of specificmetabolites is not possible.

In a preferred embodiment, imaging sequences are used that will make useof multi- echoes to code for the frequency information. Sequences thatcan produce separate water and fat ¹H-images are for example describedin G. Glover, J Magn Reson Imaging 1991;1:521-530 and S. B. Reeder etal., MRM 51 35-45 (2004). Since the metabolites to be detected and assuch their MR frequencies are known, the approach discussed in thereferences above can be applied to directly image pyruvate, alanine andlactate and preferably pyruvate, alanine, lactate and bicarbonate andmakes more efficient use of the hyperpolarised ¹³C-MR signal, giving abetter signal quality compared to the classical spectroscopic imagingtechnique, a higher spatial resolution and faster acquisition times.

Tumour tissue is often characterised by an increased perfusion andhigher metabolic activity. The process of increasing the vascular bed,angiogenesis, is induced by cells that due to their higher metabolicneeds and/or their larger distance from a capillary are not able to getenough substrates that can provide the energy needed to sustain energyhomeostasis. It is in this area, where cells have problems in producingenough energy, a marked change in metabolic pattern is expected. Tissuewith problems sustaining energy homeostasis will alter its energymetabolism which in particular results in an increased lactateproduction. Surprisingly, it is possible to make this change inmetabolism visible using hyperpolarised ¹³C-pyruvate within the short MRimaging time window available, i.e. using the high ¹³C-lactate signal inthe tumour area to discriminate the tumour from healthy tissue. As theperfusion is heterogeneous in tumour tissue, it is preferred to correctthe 13C-lactate signal for the amount of pyruvate (¹³C-pyruvate signal)available in the same region. This will allow for emphasising regions inthe tissue with a relative high lactate signal with respect to thepyruvate signal and thus improve the discrimination between tumourtissue and healthy tissue.

To correct for the pyruvate signal, both lactate and pyruvate images arenormalized to the maximum value in each individual image. Second, thenormalized lactate image is multiplied by the inverted pyruvate image,e.g. the maximum pyruvate signal in the image minus the pyruvate levelfor every pixel. As a last step, the intermediate result gained in theoperation above is multiplied by the original lactate image.

To emphasise regions with altered metabolism, the high ¹³C-lactatesignal in connection with a reduced ¹³C-alanine signal can be used in asimilar operation as described in the paragraph above. Surprisingly, theidentification of the tumour area, i.e. the discrimination betweentumour tissue and healthy tissue is improved by this correction as well.To correct for the alanine signal, both lactate and alanine images arenormalized to the maximum value in each individual image. Second, thenormalized lactate image is multiplied by the inverted alanine image,e.g. the maximum alanine signal in the image minus the alanine level forevery pixel. As a last step, the intermediate result gained in theoperation above is multiplied by the original lactate image. In asimilar manner, the ¹³C-bicarbonate signal may be included in theanalysis as well. Further a proton image acquired with our without aproton MRI contrast agent may be included in the analysis to obtainanatomical and/or perfusion information.

In another preferred embodiment, the composition according to theinvention is administered repeatedly, thus allowing dynamic studies.This is a further advantage of the composition in comparison to other MRimaging agents which, due to their relatively long circulation in thepatient's body, do not allow such dynamic studies.

The composition according to the invention is further useful as animaging agent for in vivo MR tumour staging. The same metabolic imagesand/or metabolic ratio images as described in the preceding paragraphsmay be used for this purpose with appropriate cut off categories defineddependent on tumour size and metabolic activity.

Further, the composition according to the invention is useful as animaging agent for in vivo MR tumour therapy monitoring, e.g. bymonitoring direct changes in metabolism pattern of tumours upontreatment with therapeutic antitumour agents and/or radiation treatmentor in connection with any type of interventional techniques with orwithout any kind of ablation, i.e. chemical ablation combined with radiofrequencies, microwaves or ultrasound.

Tumour MR imaging can be influenced and improved by preparing thepatient or the animal in a way that will perturb the protein metabolism,lipid metabolism or energy metabolism in general. Ways to achieve thisare known in the art, e.g. by abrosia (for instance over night), glucoseinfusion and the like.

In a preferred embodiment, the composition according to the invention isuseful as an imaging agent for in vivo MR tumour imaging, tumour therapymonitoring and tumour staging of brain tumours, breast tumours,colon/colo-rectal tumours, lung tumours, kidney tumours, head and necktumours, muscle tumours, gastric tumours, esophageal tumours, ovariantumours, pancreas tumours and prostate tumours. It has further beenfound that the composition according to the invention is especiallyuseful as an imaging agent for in vivo MR prostate tumour imaging, i.e.prostate tumour diagnosis and/or prostate tumour staging and/or prostatetumour therapy monitoring.

When a man presents to the doctor with symptoms of urinary pain ordiscomfort, prostate cancer is suspected. If the man is over 50 years, aProstate Specific Antigen (PSA) test is performed. Prostate cancer issuspected on the basis of an elevated PSA and/or abnormal Digital RectalExamination (DRE). If the PSA test is positive, the patient is sent to aspecialist (an urologist) for diagnosis using ultrasound guided biopsy.Of the two million biopsy procedures per year performed in the US andEurope, 5 out of 6 and 2 out of 3 are negative, respectively. Whendetected at an early stage, the five-year survival rate for thesepatients is 100%. As prostate cancer is the most common cancer and thesecond leading cause of cancer death in men, there is a strong medicaldemand for a method for the diagnosis of prostate tumours which iscapable of detecting prostate tumours at an early stage and which couldhelp to reduce the number of biopsy procedures.

The ¹³C-imaging of the prostate requires a transmit-receive volume¹³C-RF-coil, preferably, a transmit volume ¹³C-RF-coil in combinationwith a MR receive only endorectal RF-coil is used and more preferably, atransmit-receive phased array volume ¹³C-RF-coil in combination with aMR receive only endorectal ¹³C-RF-coil is used. Especially preferred arecoils that make the acquisition of a ¹H-prostate image possible afterthe ¹³C-imaging.

Another aspect of the invention is a composition comprising ¹³C-pyruvicacid and/or ¹³C-pyruvate and the radical of formula (I).

In a preferred embodiment, said composition comprises a radical offormula (I) where M represents hydrogen or one equivalent of aphysiologically tolerable cation. Preferably, M represents hydrogen oran alkali cation, an ammonium ion or an organic amine ion, for instancemeglumine. Most preferably, M represents hydrogen or sodium.

In a further preferred embodiment, said composition comprises a radicalof formula (I) where R1 is the same or different and representshydroxymethyl or hydroxyethyl. In another preferred embodiment, R1 isthe same or different and represents a straight chain or branchedalkoxylated C₁-C₄-hydrocarbon group, preferably —CH₂—O—(C₁-C₃-alkyl),—(CH₂)₂—O—CH₃ or —(C₁-C₃-alkyl)—O—CH₃. In another preferred embodiment,R1 is the same or different and represents a straight chain or branchedalkoxylated C₁-C₄-hydrocarbon group carrying a terminal hydroxyl group,preferably —CH₂—O—C₂H₄OH or —C₂H₄—O—CH₂OH. In a more preferredembodiment, R1 is the same and represents a straight chain alkoxylatedC₁-C₄-hydrocarbon group, preferably methoxy, —CH₂—OCH₃, —CH₂—OC₂H₅ or—CH₂—CH₂—OCH₃, most preferably —CH₂—CH₂—OCH₃.

In a particularly preferred embodiment, said composition comprises aradical of formula (I) where M represents hydrogen or sodium and R1 isthe same and represents —CH₂—CH₂—OCH₃.

In a further preferred embodiment, said composition comprises ³C-pyruvicacid and/or ¹³C-pyruvate with an isotopic enrichment of at least 75%,more preferably at least 80% and especially preferably at least 90%, anisotopic enrichment of over 90% being most preferred. Ideally, theenrichment is 100%. ¹³C-pyruvic acid and/or ¹³C-pyruvate may beisotopically enriched at the C1-position, at the C2-position, at theC3-position, at the C1- and C2-position, at the C1- and C3-position, atthe C2- and C3-position or at the C1-, the C2- and the C3-position, withthe C1-position being the preferred one.

In a particularly preferred embodiment, said composition comprises¹³C-pyruvic acid and the radical of formula (I) where M representshydrogen or sodium and R1 is the same and represents —CH₂—CH₂—OCH₃, mostpreferably said composition contains ¹³C-pyruvic acid and the radical offormula (I) where M represents hydrogen or sodium and R1 is the same andrepresents —CH₂—CH₂—OCH₃.

The compositions according to the invention comprising ¹³C-pyruvic acidand/or ¹³C-pyruvate and the radical of formula (I) are particularlyuseful for the production of hyperpolarised ¹³C-pyruvate, for instancefor the production of hyperpolarised ¹³C-pyruvate according to themethod of the invention. Hence another aspect of the invention is theuse of a composition comprising ¹³C-pyruvic acid and/or ¹³C-pyruvate andthe radical of formula (I) for the production of hyperpolarised¹³C-pyruvate.

The radicals of formula (I) where M represents hydrogen or sodium and R1is the same and represents —CH₂—CH₂—OCH₃ were found to be particularlyfavourable for use in the method according to the invention due to thefollowing properties: they are soluble in ¹³C-pyruvic acid and stablewhen dissolved therein. They further show high polarisation efficiencyin step b) of the method according to the invention and are stableduring the dissolution step c), also when a base is used in this step.They can easily be removed in step d) of the method of the invention byfor instance filtration using a hydrophobic filter material.

Those radicals are new, hence another aspect of the invention areradicals of formula (I) where M represents hydrogen or sodium and R1 isthe same and represents —CH₂—CH₂—OCH₃.

The radicals of the invention may be synthesized as described inExample 1. Briefly, the radicals may be synthesized by reacting threemolar equivalents of a metallated monomeric aryl compound with one molarequivalent of a suitably protected carboxylic acid derivative to form atrimeric intermediate. This intermediate is metallated and subsequentlyreacted with e.g. carbon dioxide to result in a tri-carboxylic tritylcarbinol which, in a further step, is treated with a strong acid togenerate a triarylmethyl cation. This cation is then reduced to form thestable trityl radical.

Yet a further aspect of the invention is the use of the radicalsaccording to the invention as a paramagnetic agent for thehyperpolarisation of compounds in a DNP process.

EXAMPLES Example 1 Synthesis ofTris(8-carboxy-2,2,6,6-(tetra(methoxyethyl)benzo-[1,2-4,5′]bis-(1,3)dithiole-4-yl)methylSodium Salt

10 g (70 mmol)Tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)methylsodium salt which had been synthesized according to Example 7 ofWO-A1-98/39277 were suspended in 280 ml dimethylacetamide under an argonatmosphere. Sodium hydride (2.75 g) followed by methyl iodide (5.2 ml)was added and the reaction which is slightly exothermic was allowed toproceed for 1 hour in a 34° C. water bath for 60 min. The addition ofsodium hydride and methyl iodide was repeated twice with the sameamounts of each of the compounds and after the final addition, themixture was stirred at room temperature for 68 hours and then pouredinto 500 ml water. The pH was adjusted to pH>13 using 40 ml of 1 M NaOH(aq) and the mixture was stirred at ambient temperature for 15 hours tohydrolyse the formed methyl esters. The mixture was then acidified using50 ml 2 M HCl (aq) to a pH of about 2 and 3 times extracted the ethylacetate (500 ml and 2×200 ml). The combined organic phase was dried overNa₂SO₄ and then evaporated to dryness. The crude product (24 g) waspurified by preparative HPLC using acetonitrile/water as eluents. Thecollected fractions were evaporated to remove acetonitrile. Theremaining water phase was extracted with ethyl acetate and the organicphase was dried over Na₂SO₄ and then evaporated to dryness. Water (200ml) was added to the residue and the pH was carefully adjusted with 0.1M NaOH (aq) to 7, the residue slowly dissolving during this process.After neutralization, the aqueous solution was freeze dried.

Example 2 Production of Hyperpolarised ¹³C-Pyruvate using ¹³C-PyruvicAcid and the Radical of Example 1

A 20 mM solution was prepared by dissolving 5.0 mg of the radical ofExample 1 in ¹³C₁-pyruvic acid (164 μl). The sample was mixed tohomogeneity and an aliquot of the solution (41 mg) was placed in asample cup and inserted in the DNP polariser.

The sample was polarised under DNP conditions at 1.2 K in a 3.35 Tmagnetic field under irradiation with microwave (93.950 GHz). After 2hours the polarisation was stopped and the sample was dissolved using adissolution device according to WO-A-02/37132 in an aqueous solution ofsodium hydroxide and tris(hydroxymethyl)-aminomethane (TRIS) to providea neutral solution of hyperpolarized sodium¹³C₁-pyruvate. The dissolvedsample was rapidly analysed with ¹³C-NMR to assess the polarisation anda 19.0 % ¹³C polarisation was obtained.

Example 3 Production of Hyperpolarised ¹³C-Pyruvate using ¹³C-PyruvicAcid and the Radical of Example 1

A 15 mM solution was prepared by dissolving the radical of Example 1(209.1 mg) in a mixture of ¹³C₁-pyruvic acid (553 mg) and unlabelledpyruvic acid (10.505 g). The sample was mixed to homogeneity and analiquot of the solution (2.015 g) was placed in a sample cup andinserted in the DNP polariser.

The sample was polarised under DNP conditions at 1.2 K in a 3.35 Tmagnetic field under irradiation with microwave (93.950 GHz). After 4hours the polarisation was stopped and the sample was dissolved using adissolution device according to WO-A-02/37132 in an aqueous solution ofsodium hydroxide and tris(hydroxymethyl)aminomethane (TRIS) to provide aneutral solution of hyperpolarized sodium ¹³C₁-pyruvate with a totalpyruvate concentration of 0.5 M in 100 mM TRIS buffer. In series withthe dissolution device a chromatographic column was connected. Thecolumn consists of a cartridge (D=38 mm; h=10 mm) containing hydrophobicpacking material (Bondesil-C18, 40UM Part #:12213012) supplied byVarian. The dissolved sample was forced through the column whichselectively adsorbed the radical. The filtered solution was rapidlyanalysed with ¹³C-NMR to assess the polarisation, 16.5% ¹³C polarisationwas obtained. The residual radical concentration was subsequentlyanalysed with a UV spectrophotometer at 469 nm and was determined to bebelow the detection limit of 0.1 μM.

Example 4 Production of Hyperpolarised ¹³C-Pyruvate using ¹³C-PyruvicAcid and Tris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl)methyl Sodium Salt

Tris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo[1,2-d:4,5-d′]-bis-(1,3)-dithiole-4-yl)methylsodium salt was synthesised as described in Example 29 in WO-A-97/09633.

A 20 mM solution was prepared by dissolvingTris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo[1,2-d:4,5-d′]-bis-(1,3)-dithiole-4-yl)methyl sodium salt in¹³C₁-pyruvic acid (83.1 mg). The sample was mixed to homogeneity, placedin a sample cup and inserted in the DNP polariser. The sample waspolarised under DNP conditions at 1.2 K in a 3.35 T magnetic field underirradiation with microwave (93.950 GHz). The ¹³C-NMR signal from thesample was acquired using a Varian Inova-200 NMR spectrometer. The DNPenhancement was calculated from a measurement of the thermal equilibrium¹³C-NMR signal and the enhanced NMR signal. 16% ¹³C polarisation wasobtained.

Example 5 Tumour Imaging Using Hyperpolarised ¹³C-Pyruvate as ImagingAgent

5.1 Tumour animal model and tumour preparation

R3230AC is a rat mammary adenocarcinoma that can be maintained in femaleFischer 344 rats. To establish the animal tumour model, a frozen vial ofR32030 cells containing RPMI 1640, 10% FBS and 10% DMSO was rapidlythawed in 37° C. Thereafter, the cell solution was transferred to FBSand increasing volumes of RPMI 1640 were added. Finally, the cellsuspension was transferred to a 25 cm² growth flask and put into anincubator at 37° C., 5% CO₂. Growth media were changed every other day.At the day of rat infection, removal of cells was carried out either bymechanical force or by means of trypsin. Cells were washed usingphosphate buffer lacking calcium and magnesium. Trypsin (0.05% trypsinin 0.02% EDTA) was added for 2-5 min. Then, 5 ml FBS was added and thecells were transferred into a beaker containing RPMI 1640 with FCS andantibiotics (100 IU/ml penicillin, 100 IU/ml streptomycin and 2.5 μg/mlamphotericin B). The cell solution was centrifugated and the cell pelletwas resuspended in 20 ml RPMI with FBS and antibiotics, centrifugationand resuspension was repeated. The cells were then aliquoted to vialscontaining 4×10⁶ cells/ml RPMI 1640. To obtain donor tumours, femaleFischer 344 rats (Charles River, 180-200 g) were anaesthetised and 0.3ml of the cell suspension was subcutaneously injected in the inguinalregion on both sides. 15 and 22 days later, pieces of tumour wereprepared as described in F. A. Burgener et al., Invest Radiol 22/6(1987), 472-478; S. Saini et al., J. Magn. Reson. 129/1 (1997), 35-43).Two incisions were made on the ventral abdomen of recipient femaleFischer rats. A tumour piece was inserted into each pocket and theincisions were closed. Rats were brought to imaging 12-14 days aftertumour engrafting.

5.2 Rat Preparation and Proton MR Imaging

Weighed rats were anaesthetised using isoflurane (2-3%) and kept on aheated table to ensure a body temperature of about 37° C. A catheter wasintroduced into the tail vein and into the arteria carotis communissinistra. The rats were transported to the MR machine and placed on ahome-built pad that was heated to approx. 37° C. by means of circulatingFC-104 Fluorinert. This liquid will not give rise to background signalsin ¹H- and ¹³C-MR imaging. Anaesthesia was continued by means of 1-2%isoflurane delivered via a long tube to an open-breathing system at arate of 0.4 L/min. The arterial catheter was connected via a T-tube to apressure recorder and a pump delivering saline (rate 0.15 L/min) toprevent catheter clotting. Rats were positioned in a rat MR coil (RapidBiomedical, Germany) and imaging using a standard proton MR imagingsequence to get anatomical information and to determine the location ofthe tumour.

5.3 ¹³C-MR Imaging

Based on the proton frequency found by the MR system the MR frequencyfor ¹³C₁-alanine was calculated according to the following equation:

Frequency ¹³C₁-alanine=0.25144×[(system frequencyproton×1.00021)−0.000397708]

The frequency calculated positioned the MR signal arising from¹³C₁-alanine on resonance with ¹³C₁-lactate on the left and¹³C₁-pyruvate resonating on the right of ¹³C₁-alanine. An unlocalised MRspectroscopy sequence was run to ensure that the 13C-MR coil and thesystem MR frequency had been set up correctly. The ¹³C-image locationwas positioned to cover the tumour (slice thickness 10 mm, in planepixel size 5×5 mm²). In the reconstruction phase, the image data waszero-filled to result in 2.5×2.5×10 mm³ resolution. ¹³C₁-pyruvate inTRIS buffer (90 mM) was injected in a dose of 10 ml/kg during a periodof 12 s with a minimum volume of 2 ml into the tail vein and 30 s afterthe start of the injection (i.e. 18 s after finishing the injection),the chemical shift ¹³C-MR sequence was started.

5.4 Analysis of the MR Imaging Data

MR imaging resulted in a matrix containing 16×16 elements in which eachelement or voxel/pixel contains a ¹³C-MR spectrum. In the reconstructionphase, the matrix was zero-filled to 32×32, a mathematical operationthat helps to improve the spatial resolution. The dataset to be analysedcontained 1024 spectra as was exported to Dicom® format (DICOM is theregistered trademark of the National Electrical ManufacturersAssociation for its standards publications relating to digitalcommunications of medical information) for further analysis. About halfof these spectra did not contain MR signals as the position of thesevoxels was outside the animal. A location within the animal revealedvoxels with high pyruvate signals and negligible lactate and alaninesignal (blood pool) while other voxels showed pyruvate, alanine andlactate in about equal intensity.

The amplitudes for pyruvate, alanine and lactate were estimated usingtime domain fitting procedures which included the following: the zeroorder phase is constant over the dataset, the first order phase is 1.4ms, the line width or damping in the time domain is allowed to varybetween 0.5 and 3 times the average line width of the whole dataset foreach metabolite independently and the frequency is allowed to vary with20 Hz in both directions with respect to the average frequency foundover the whole dataset for the highest peak, which has to be identifiedby the user.

The amplitudes for lactate, alanine and pyruvate were reordered in amatrix and resampled to match the resolution of the proton anatomical MRimage. The ¹³C-MR images were projected on the anatomical images usingan automated procedure to obtain an operator-independent result. Theresults were displayed in image sets containing the anatomical protonimage of the tumour in the rat, the metabolic ¹³C-image for pyruvate,lactate and alanine projected onto the anatomical image, images showingfor every pixel

-   -   a)        ([lactate]_(norm)×([pyruvate]_(max)−[pyruvate])_(norm))×[lactate]        and    -   b)        ([lactate]_(norm)×([alanine]_(max)−[alanine])_(norm))×[lactate]        in which the term “[. . .]norm represents the normalised        amplitude, i.e. scaled to its highest value in the metabolic        image and [lactate] the amplitude calculated.

A successful result for the discrimination of tumour tissue and healthytissue in a metabolic ¹³C-MR image was defined as highest lactate signalin the tumour area or a high weighted ratio lactate over pyruvate in thetumour area and a high weighted lactate over alanine ratio in the samepixel location.

5.5 Biological Analysis

Tumour sites were visually inspected to detect signs of bleeding.Tumours were liberated from the rat bodies, weighed and cut in half.Tumour interiors were inspected visually assessing homogeneity, necrosisand bleeding. The tumour tissues were stored in 4% formalin.

A tumour-bearing rat was considered to be appropriate for evaluation ifthe following criteria were met: tumour weight>100 mg, no visiblenecrosis or cysts in the tumour interior, a body temperature above 35°C. and a mean arterial blood pressure above 60 mm Hg at time of MRinvestigation.

5.6 Results

In total 30 different tumours were imaged in 18 rats. 1 rat failed and 3tumours failed the biological criteria described in the precedingparagraph 5.5. The remaining 26 tumours in 17 rats were homogenous andhad a massive non-necrotic interior. The average polarisation of¹³C₁-pyruvate at the time of injection was 21.2±2.9% (mean ±SD) and thepH was 8.08±0.14 (mean±SD).

FIG. 1 displays a typical set of images of one imaged rat with (1) theproton reference image, wherein the arrows indicate the tumourlocations, (2) the ¹³C-pyruvate image, (3) the ¹³C-lactate image (4) the¹³C-alanine image (5) the ¹³C-lactate image corrected for ¹³C-pyruvateand (6) the ¹³C-lactate image corrected for ¹³C-alanine. Images (2) to(6) are fused with the proton reference image.

FIG. 2 displays the same set of images, however with images (2) to (6)which are not fused with the anatomical proton image.

As a result, tumour location is indicated by a high pyruvate signal (2),due to high metabolic activity. However the lactate signal (3)ultimately identifies the correct location of the tumour. Alanine isvisible in the skeletal muscle and is absent in the tumour tissue (4).The pyruvate and alanine corrected lactate images (5) and (6) result inan excellent contrast for the tumour as well.

It was thus demonstrated that the tumour location in the metabolicimages is indicated by a high lactate signal, a high lactate signalcorrected for pyruvate and a high lactate signal corrected for alanine.

The analysis of the metabolic ¹³C-MR images revealed a metaboliccontrast in the tumour area in

-   -   24 out of 26 tumours for the lactate signal    -   26 out of 26 tumours for the lactate signal, pyruvate corrected        (5.5, a))    -   26 out of 26 tumours for the lactate signal, alanine corrected        (5.5, b))

The overall rate of success for this study was 26 out of 26, or 100%.

With this study, it was demonstrated that the hyperpolarised¹³C₁-pyruvate reach the region of interest (tumour) in a time periodwhich makes it possible to image the compound, that the compound and itsmetabolites can be imaged and that metabolic contrast can be obtained.

1. A method for the discrimination between healthy and tumour tissue,said method comprising (a) acquiring direct ¹³C-MR images of¹³C-pyruvate and its ¹³C-containing metabolites alanine, lactate andoptionally bicarbonate from a subject pre-administered with acomposition comprising hyperpolarised ¹³C-pyruvate, (b) optionallycorrecting the lactate signal for the amount of pyruvate and/or alanineto obtain a weighted lactate over pyruvate and/or lactate over alanineimage, wherein tumour tissue in said ¹³C-images is indicated by thehighest lactate signal and/or, if the correction in step (b) has beencarried out, by a high weighted lactate over pyruvate and/or lactateover alanine signal.
 2. A method according to claim 1 wherein thehyperpolarised ¹³C-pyruvate is obtained by hyperpolarising at least oneof ¹³C-pyruvic acid and 13C-pyruvate by the DNP method.
 3. A methodaccording to claim 1 wherein the composition comprising ¹³C-pyruvatefurther comprises one or more buffers selected from the group consistingof phosphate buffer (KH₂PO₄/Na₂HPO₄), ACES, PIPES, imidazole/HCl, BES,MOPS, HEPES, TES, TRIS, HEPPS and TRICIN.
 4. A method according to claim1 wherein imaging sequences that make use of multiechoes to code forfrequency information are used for acquiring the direct ¹³C-images instep a).
 5. A method according to claim 1 wherein the direct ¹³C-imagesin step a) are acquired at less than 400 s after the administration ofthe composition comprising ¹³C-pyruvate.
 6. A method according to claim1 further comprising the step of acquiring a proton image with orwithout a proton MRI contrast agent.
 7. A method according to claim 1wherein step b) further comprises correcting the lactate signal for theamount of bicarbonate to obtain a weighted lactate over bicarbonateimage and, wherein if correction in step b) has been carried out, tumourtissue in said 13C-images is indicated by the highest lactate signal, bya high weighted lactate over pyruvate and/or lactate over alanine and/orlactate over bicarbonate signal.
 8. A method according to claim 1wherein step b) is mandatory.
 9. A method according to claim 8 whereinsaid correction is carried out by (i) normalizing the lactate andpyruvate and/or alanine and/or bicarbonate images to the maximum valuein each individual image (ii) multiplying the normalized lactate imageby the inverted pyruvate and/or alanine and/or bicarbonate image; and(iii) multiplying the results of step (ii) by the original lactateimage.
 10. A method according to claim 1 wherein the tumour is a braintumour, breast tumour, colon tumour, lung tumour, kidney tumour, headand neck tumour, muscle tumour, ovarian tumour, gastric tumour,pancreatic tumour, esophageal tumour or prostate tumour.
 11. A methodaccording to claim 1 for in vivo MR tumour therapy monitoring and/ortumour staging.